1. Field of the Invention
The present invention relates to a complement dependent bactericidal assay useful for the measurement of complement, bactericidal properties of serum, and immune components of serum.
2. Brief Description of the Prior Art
A complex of proteins, which is known as complement and is abbreviated as C', is present in the fresh serum of vertebrates, is bound by antigen/antibody aggregates, and plays an important auxiliary role in antigen/antibody interactions and in host defense. Complement consists of nine major components and nine or more other components that react in a known sequence when antigen/antibody combine. C' reacts at the sites of antigen/antibody combination on cell membranes to cause lysis. In the case of erythrocytes this causes the release of hemoglobin into solution, an effect which is called hemolysis.
Certain gram-negative bacteria coated with specific antibody can be lysed by C' acting through the same reaction sequence as in red cell lysis. Gram-positive bacteria and mycobacteria, however, do not appear to be susceptible to the lytic reaction of complement, although their resistance is not well understood. The lysis of gram-negative bacteria by C' is known as the bactericidal effect of complement. (See, for example, Eisen, "Immunology. An Introduction to Molecular and Cellular Principles of the Immune Responses," 2nd edition, Harper & Rowe Publishers, Inc., 1974, pp. 513-523; and Kabat, "Structural Concepts in Immunology and Immunochemistry," Holt, Reinhardt & Winston, Inc., 1968, pp. 46-48).
The hemolytic properties of complement have been used in the complement fixation assay, an important laboratory procedure for detecting and measuring many different kinds of antigens and antibodies. Thus, if sheep erthyrocytes are optimally coated with non-agglutinating amounts of antibodies to the cells, the addition of C' and the presence of adequate concentration of Mg++ and Ca++ ions promptly causes the cells to lyse. The extent of lysis is evaluated qualitatively by inspection, or quantitatively by determining the concentration of supernatant hemoglobin after sedimentation of intact cells and stroma. These methods have been utilized to measure immune components of serum, for example by Molinaro et al, U.S. Pat. No. 4,130,634 and Bartos et al, U.S. Pat. No. 4,239,746, which are herein incorporated by reference.
A general test for the assay of antibodies and antigens, albeit without using complement, has been described by Young, U.S. Pat. No. 4,104,126, wherein an antigen is conjugated with a bacteriophage, and the conjugate allowed to compete with the antigen in the specimen under assay for a number of binding sites on antibody. The phage conjugates surviving antibody inactivation are quantified by determining intracellular constituents of host bacteria subsequently infected by the bacteriophage remaining viable, and which can be related to the levels of antigen originally present in the specimen. For example, a colorimetric assay for .beta.-galactosidase freed by phage lysis of E. coli is described in Young (column 5), and the possibility is mentioned of increasing the released enzyme by induction.
The complement hemolytic assay is somewhat limited in its approach in that it always requires sheep red blood cells and it does not measure bacteriolysis, thereby being meaningless in assessing the patient's natural defenses against infection. A bacteriolytic assay such as that of Young, also fails to determine the total bacteriolytic properties of the serum itself, since bacteriolysis is carried out with an externally added conjugate containing phage.
Therefore a need continues to exist for an efficient, safe and sensitive assay for the detection of the bacteriolytic properties of a sample and measurement of other immune components.